There are several analytical methods for measuring characteristics of cells and cell-like materials (such as liposomes) which require the use of fresh, live cells for standardization of the equipment. The most important of these methods is flow cytometry, the measurement of cells in a moving liquid stream, which is a valuable analysis tool in research laboratories. In the apparatus used in cytometry, called a cytometer or flow cytometer, a fluid flows through a sensing orifice in the presence of an electric field. If the particle enters the orifice it will disturb the electric field, increasing the resistance of the circuit and the size of the disturbance will be proportional to the volume and relative particle size to the volume of the orifice. An electric pulse is caused as each particle passes through the orifice, and the pulse height will be proportional to the size of the particle. Using sophisticated focusing techniques to enhance the resolution, the flow cytometer can be used for electrical sizing of the cells. Furthermore, if the cells are chemically tagged with fluorescent molecules when they are flowed past a source of monochromatic light (a laser), a lens system can be used to detect the amount of light scattered as well as the fluorescent signals. The scattered light intensity is a complex function of cell size and refractive properties of the cell but generally it can be used as a way of measuring the cell area. The fluorescent signals are roughly a measure of the concentration of the fluorescent molecule in a cell or on its surface. The wide angle scatter of the light can be measured as a tool for distinguishing cells of equal size which have different internal refraction properties. For example, cells with more internal structure such as granulocytes, will scatter more light at right angles than will lymphocytes. Therefore, the flow cytometer is an instrument for rapid and accurate measurement of particle volume (electrical resistance sizing), particle area (forward light scatter), fluorescent atom concentration (fluorescent emission), and particle granularity (right angle light scatter). If a charging pulse is applied to the stream, cells can be selectively deflected into distinct streams and the cells in those streams counted. In this way the flow cytometer is also a tool for sorting and counting cells.
The present invention is directed to providing materials for calibrating cell-analyzing apparatus such as a flow cytometer. The accuracy of the flow cytometer is directly related to the ability to calibrate the system with calibrating particles which should be highly stable, readily available particles of known size and light scattering characteristics. One of the calibrating standards typically used is a plastic bead, but in many instances these are unsatisfactory since a solid sphere (which does not deform) displaces additional current at its leading and trailing edge. As a result an electrical sizing system will over estimate the size of a plastic sphere, usually by a factor of about 1.5.
If one is to use the flow cytometer to measure live cells, then it must be calibrated with live cells, since a live cell has a different index of refraction than a dead cell. Plastic beads also lack the cell surface antigens which are often measured in blood particles such as lymphocytes. Fluorescence is also difficult to calibrate, since the calibrating particle can degrade from leaching or bleaching of the dye. Accordingly, when used for identifying cell subpopulations, some subpopulations are identified without the use of cell-specific chemical markers. For example, in blood leukocyte populations containing lymphocytes, monocytes and granulocytes, measurements of light scattered by cells at two different angles (commonly called the forward scatter and orthogonal or wide angle scatter) from an incident laser beam can discriminate the three cell types sufficiently for differential leukocyte counting in clinical hematology. With the added use of cell specific chemical markers, such as fluorescent markers, other characteristics of the individual cell subpopulations may be determined by a gated analysis, such as the CD4:CD8 subpopulation of lymphocytes.
Cell standards include polystyrene particles in ranges of sizes and colors consisting of UV-excited blue fluorescent and green-excited orange fluorescent spheres in addition to green fluorescent spheres.
For standardizing immunofluorescent measurements, fixed animal thymocytes labeled with covalently attached dyes have been used. Chicken and rainbow trout erythrocytes have also been used as standards for DNA content estimations (Vindelov, et al., Cytometry 3:328 (1982)). Glutaraldehyde-fixed chicken erythrocytes have also been used since they fluoresce without staining.
U.S. Pat. No. 5,059,518 to Kortright, et al. discloses the lyophilization of mammalian cells as control cells for immunoassays and other hematological measurements. The cells are lyophilized in a solution of trehalose and are rehydrated for use. Trehalose, a disaccharide, does not permeate the cell membranes during lyophilization.
The invention provides further improvement over the art for storage of cells. Hospitals must keep samples of rare type cell standards on hand (such as AB- and rare genotype standards) which are more expensive and difficult to acquire than commonly available cell standards. These must be replaced within the three to four week effective shelf life if they are not used.
Even during the seven weeks of refrigeration of red blood cells in Alsever's suspensions, key metabolites such as ATP, are depleted and slow cell lysis occurs.
There is therefore a need for providing blood samples which have longer refrigerated (or room temperature) storage lives than the refrigerated Alsever's red cell suspensions.
The present invention provides frozen or freeze-dried (lyophilized) cells, including erythrocytes and lymphocytes, having the advantages of convenient dry storage in a ready-to-use format. These cells, when reconstituted, are useful as standards for cytometry.
It is yet another object of the present invention to provide frozen or freeze-dried lymphocytes, monocytes and granulocytes which, when reconstituted, preserve key light scattering, staining ability, particularly for CD4:CD8 ratios.
It is an object of the present invention to provide cell standards for analytical methods by using lyophilized red blood cells, lymphocytes, monocytes or granulocytes.
It is another object of the present invention to provide instrument calibration and quality control standards for flow cytometry.
The present invention permits dry storage of intact cells which after rehydration retain key characteristics to provide cell standards.